Molecular mechanisms of TRPV5 gating

项目摘要/摘要 肾结石，通常被称为肾结石，是一种常见的疾病，影响大约30% 百万美国人。肾结石形成最关键的危险因素之一是高钙尿症。 尿钙水平(钙离子)。瞬时受体电位香草型5(TRPV5)通道，即 主要在肾脏表达，已被发现对血液中重新吸收钙离子是必不可少的。损失 或TRPV5功能障碍已被证明可严重升高尿钙水平和肾脏的发生 石头。TRPV5位于肾单位上皮细胞的顶膜，可使细胞内的钙离子重吸收。 尿液沿其浓度梯度。在没有调制器的情况下，TRPV5已被提议 天生活跃的。内源性调节剂，如钙调素(CaM)和磷脂酰肌醇4，5- 双磷酸盐(PI(4，5)P2)分别以封闭构象和开放构象稳定TRPV5。 蛋白激酶C(PKC)介导的TRPV5在Ser299和Ser654残基的磷酸化已被证明是 提高信道的开放概率。像益康唑和咪康唑这样的小分子抗真菌药物已经被 证明能抑制TRPV5。然而，这种通道调制和选通的分子细节很差 明白了。因此，这项提议的目标是将冷冻电子显微镜(CRYO-EM)与 用生化、电生理学和计算方法来揭示 TRPV5门控。在原子水平深入研究TRPV5将为靶向药物铺平道路 控制和治疗高钙尿和肾结石的发现。

PROJECT SUMMARY/ABSTRACT Nephrolithiasis, generally known as kidney stones, is a common disease that affects approximately 30 million Americans. One of the most critical risk factors for kidney stone formation is hypercalciuria, or high levels of urine calcium (Ca2+). The transient receptor potential vanilloid type 5 (TRPV5) channel, which is primarily expressed in the kidney, has been found to be essential for reabsorption of Ca2+ into the blood. Loss or dysfunction of TRPV5 has been shown to severely increase urine Ca2+ levels and the occurrence of kidney stones. TRPV5 is found in the apical membrane of the nephron epithelium and allows Ca2+ reabsorption from the urine along its concentration gradient. In the absence of modulators, TRPV5 has been proposed to be constitutively active. Endogenous modulators such as calmodulin (CaM) and phosphatidylinositol 4,5- bisphosphate (PI(4,5)P2) have been found to stabilize TRPV5 in the closed or open conformation, respectively. Protein kinase C (PKC)-mediated TRPV5 phosphorylation at Ser299 and Ser654 residues has been shown to enhance open probability of the channel. Small molecule antifungals like econazole and miconazole have been demonstrated to inhibit TRPV5. However, molecular details of this channel modulation and gating are poorly understood. Therefore, the goal of this proposal is to utilize cryo-electron microscopy (cryo-EM) in conjunction with biochemical, electrophysiological and computational approaches to uncover the molecular mechanisms of TRPV5 gating. An in-depth investigation of TRPV5 at the atomic level will pave the way for targeted drug discovery for the control and treatment of hypercalciuria and nephrolithiasis.